PROJECT SUMMARY SLC4 proteins in the kidney play an essential role in mediating the coupled transport (symporters, exchangers) of Na+, HCO3?, CO32?, and Cl? in the proximal tubule and collecting duct. Infants and children with mutations in the electrogenic Na+-CO32? symporter NBCe1 and the Cl?/HCO3? exchanger AE1 develop severe proximal and distal renal tubular acidosis respectively. How disease causing mutations impair their transport mechanisms at the molecular level is unknown. Towards this goal, we have recently solved by cryoelectron microscopy (cryoEM) the outward facing structure of NBCe1, the inward and outward facing structures of AE1, and have obtained 2D class averages of the Na+-CO32?/Cl? exchanger NDCBE representing major advances in the field. These significant advances coupled with our preliminary functional mutagenesis and Molecular Dynamics computational analyses make it possible for the first time in the transport field to achieve the long-term objective of understanding the detailed structure-functional properties of SLC4 transporters and their impairment by disease causing mutations. To accomplish this objective, the project addresses the following specific aims: Aim 1: Structural Determinants of the AE1, NBCe1, and NDCBE Ion Coordination Sites, Transport Modes and Ion Specificities: In this aim, using cryoEM, functional mutagenesis and Molecular Dynamics computational analyses, the hypothesis that the ion coordination sites in these transporters encode both their respective transport modes and unique ion transport specificities will be examined. Aim 2: Characterize the Structural Components of AE1, NBCe1 and NDCBE Permeation Pores, Ion Selectivity and Energetics: In this aim, using cryoEM, functional mutagenesis and Molecular Dynamics computational analyses, the hypothesis that differences in the structure and energetics of ion permeation among these transporters plays an important role in determining the selectivity of ions reaching their coordination sites in both outward and inward facing conformations will be examined. Aim 3: Transport Models for AE1, NBCe1 and NDCBE, and Characterization of the Transport Abnormalities Induced by Renal Tubular Acidosis Causing Mutations: The dynamic transport models of AE1, NBCe1 and NDCBE will be generated based on preliminary data, and the results obtained in Aims 1 and 2. How disease causing mutations impair these mechanistic transporter molecular models will be determined. This proposal represents a significant contribution to Nephrology and Medicine given the importance of SLC4 transporters in regulating kidney ion balance, systemic acid-base chemistry, blood pressure, and the maintenance of cell function and growth.